1. Field of the Invention
The present invention relates to molecular inhibitors of Nucleophosmin (NPM) activity. NPM is a multifunctional nucleolar phosphoprotein that is dysregulated in human malignancies leading to anti-apoptosis and inhibition of differentiation. NPM inhibitors are useful for treating diseases or disorders associated with NPM-associated cellular dysregulation, such as cancer.
2. Description of the Related Art
Nucleophosmin (NPM), also known as B23, NO38, or numatrin (Lim and Wang, 2006), is a nucleolar phosphoprotein composed of an N-terminal globular domain (1-110 residues) and a C-terminal domain (111-294 residues) rich in acidic residues. NPM was initially identified as a critical player in ribosome biogenesis (Lim and Wang, 2006). Since then a number of cellular activities associated with NPM indicate that this protein has multiple functions, especially in cell proliferation, cytoplasmic/nuclear shuttle transportation, nucleic acid binding, ribonucleic cleavage, centrosome duplication and molecular chaperoning (Okuda, 2002; Okuwaki et al., 2001; Ye, 2005). NPM shuttles between the nucleolus and the cytoplasm, and it also translocates from the nucleolus to the nucleoplasm during the stationary phase of growth or during treatment with certain antitumor drugs (Chou and Yung, 1995; Yung et al., 1990).
Under native conditions, NPM exists as an oligomer (Herrera et al., 1996; Namboodiri et al., 2004). The structural features of NPM consist of an oligomerization domain, a metal-binding motif, a bipartitenuclear localization signal, phosphorylation sites and a nucleolar localization signal (Wang et al., 1993; Wang et al., 2005). Mounting evidence supports that NPM interacts with a variety of proteins including nucleolin (Li et al., 1996), cell cycle related protein p120 (Valdez et al., 1994), HIV-1 Rev protein (Fankhauser et al., 1991), HDM2 (Kurki et al., 2004), tumor suppressor ARF (Bertwistle et al., 2004; Itahana et al., 2003; Korgaonkar et al., 2005), p53 (Colombo et al., 2002; Li et al., 2004; Maiguel et al., 2004) and pRb (Takemura et al., 1999).
NPM has been found to be more abundant in tumor and growing cells than in normal resting cells (Chan et al., 1989; Feuerstein et al., 1988). In fact, NPM has been proposed as a tumor marker for prostate (Subong et al., 1999), colon (Nozawa et al., 1996), ovarian (Shields et al., 1997), gastric (Tanaka et al., 1992), and bladder (Yeh et al., 2006) cancers because NPM expression is markedly higher in these tumor cells than in the corresponding normal cells. Notably, over-expression of NPM in NIH3T3 cells results in malignant transformation (Kondo et al., 1997). NPM expression is down-regulated in cells undergoing differentiation or apoptosis (Patterson et al., 1995; van Belzen et al., 1995). However, overexpression of NPM leads to the inhibition of apoptotic cell death (Grisendi et al., 2006; Ye, 2005). In addition, NPM is frequently found in chromosomal translocations associated with hematological malignancies (Naoe et al., 2006).
The role of NPM as an oncogene is further enhanced as it binds several tumor suppressor genes, including pRb (Takemura et al., 1999), p14ARF (Brady et al., 2004; Gjerset, 2006; Lee et al., 2005; Zhang, 2004) and p53 (Colombo et al., 2002; Li et al., 2004; Maiguel et al., 2004). The p53/p14ARF/Mdm2 (Hdm2) stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability and therapy induced DNA damage. Recently, NPM emerged as a p14ARF binding protein and negative regulator of p53 by binding the same domains that mediate nucleolar localization and Hdm2 binding. This indicates that NPM may control p14ARF localization and compete with Hdm2 for ARF association. NPM knockdown markedly enhances ARF-Mdm2 association and diminishes ARF nucleolar localization. In addition, it has been shown that NPM directly interacts with p53 and down regulates its transcriptional activity (Maiguel et al., 2004). NPM binds to the p53 N-terminal end and prevents p53 phosphorylation at Ser15 in response to low doses of UV radiation. However, down regulation of NPM by small interfering RNA allowed p53 phosphorylation to occur. Similar results were observed in hematopoietic cells following ionizing irradiation treatments (Li et al., 2005) and in lymphoblasts exposed to hypoxia (Li et al., 2004). Together these data indicate that NPM is a natural repressor of p53 that may contribute to dampening p53 function during cellular growth or in the presence of DNA damage. Hence, NPM is an oncoprotein that if selectively targeted could be a potential therapy for cancer. An example of this concept is an NPM-binding peptide derived from the Rev protein which leads to cytotoxicity in Ras-transformed NIH-3T3 (Ras-3T3) cells, as well to inhibition of tumor growth in a nude mouse model (Chan et al., 2005).
Targeting protein-protein interaction sites as potential intervention points or ‘hot spots’ for the development of anti-cancer agents has made clear inroads and is now a reality (Arkin and Wells, 2004). This is due to the discovery of several specific small molecular agents targeting anti-apoptotic targets such as Bcl-2-Bax (Oltersdorf et al., 2005), MDM2-p53 (Vassilev et al., 2004) and XIAP-SMAC (Oost et al., 2004). Under native conditions, NPM exists as oligomers via its N-terminal molecular chaperone domain (Herrera et al., 1996; Hingorani et al., 2000; Namboodiri et al., 2004).
Over-expression of NPM prevents apoptosis induced by a variety of stimuli including hypoxia, radiation and retinoic acid (Grisendi et al., 2006; Ye, 2005). NPM is rapidly upregulated after UV irradiation resulting in resistance to UV-mediated apoptosis through enhanced DNA repair (Grisendi et al., 2006). As a nuclear PI(3,4,5)P3 receptor, NPM inhibits DNA fragmentation activity of caspase-activated-DNase (CAD) and mediates an anti-apoptotic effect of nerve growth factor (NGF) (Ahn et al., 2005). In addition, NPM was found to target the interferon-inducible, double-stranded RNA-dependent protein kinase (PKR) and inhibit apoptosis (Grisendi et al., 2006). However, down regulation of NPM potentiates apoptotic cell death (Ye, 2005). A mouse NPM knockout experiment demonstrated that lack of NPM expression results in accumulation of DNA damage (Colombo et al., 2005).
Normally, the expression of cellular p53 protein is very low due to its relatively short half-life. Human MDM2 (HDM2) regulates p53 levels by binding to p53 and initiating proteasomal degradation (Oren, 1999). HDM2 also modulates several tumor suppressors, including p14ARF. Recently, NPM emerged as a p4ARF binding protein and a negative regulator of p53 by binding to the same domains that mediate nucleolar localization and HDM2 binding. This indicates that NPM may control p14ARF localization and compete with HDM2 for p14ARF association. DNA damaging treatments disrupt the p14ARF-NPM interaction and trigger a transient subnuclear redistribution of p14ARF to the nucleoplasm, where it interacts with HDM2 and prevents p53 degradation (Bertwistle et al., 2004; Gjerset, 2006; Korgaonkar et al., 2005; Lee et al., 2005). In addition, NPM has been shown to interact with the p53 N-terminal domain directly and inhibits p53
phosphorylation on Ser15 in response to low doses of UV radiation (Colombo et al., 2002; Maiguel et al., 2004). Collectively, the data indicate that NPM is a natural repressor of p53 that may contribute to dampening of p53 function during cellular growth or in the presence of low levels of DNA damage. In contrast, contradictory reports indicate that NPM stabilizes p53 by binding to HDM2 and protecting p53 from HDM2-mediated degradation (Kurki et al., 2004).
Administration of large molecule NPM inhibitors such as peptides or antibodies imposes many obstacles on treatment and makes it difficult to provide a convenient and economical pharmaceutical agent. There is a need to find simple, easily administered small molecules, such as those having molecular weights of less than 1,000 Daltons, that inhibit NPM functions associated with cellular dysregulation and malignancy. With these obstacles and goals in mind, the inventors sought out and identified particular classes of small molecules that inhibit NPM functions.